TWI717434B - Means and method for enhancing growth of fish - Google Patents

Abstract

The present invention provides an engineered plasmid, a recombinant protein, a recombinantBacillus subtilis, an animal food/feed additive, a method of making recombinantBacillus subtilis, a method of making a culture lysate, and a method of promoting growth rate of fish.

Description

Means and methods for promoting the growth of fish

Cross-reference of related applications This application claims the priority or rights of Hong Kong Patent Application No. 15112136.3 filed on December 9, 2015, which is incorporated herein by reference in its entirety.

The present invention relates to bioengineering means and methods for promoting the growth of fish.

Growth rate is considered one of the most important focuses of fish farms, because there is a direct relationship between growth performance and yield (the resulting profit). Therefore, different attempts have been made in this industry to increase the growth rate of fish.

Fish growth hormone (FSH) has been proposed to promote the growth of fish. One way to use FSH to promote fish growth is to soak the fish in a water bath in which FSH is dissolved. However, this method has strict restrictions. For example, on a large scale, using FSH in this way is inefficient, expensive, and impractical. Second, many fish cannot absorb FSH well in this way. Another limitation is that it is not clear whether it is safe to eat fish treated with this FSH.

The present invention seeks to solve the aforementioned problems, or at least provide the public with useful alternatives.

According to the first aspect of the present invention, there is provided a method for expressing a gene encoding fish growth hormone (tiGH) of Oreochromis hornorum in a B. subtilis ( B. subtilis ) host, the method comprising the following Steps:-Preparation of a recombinant DNA cassette (DNA cassette), the DNA expression cassette from 5'end to 3'end includes: three pairs- veg A promoter ( veg A) and lac operating gene ( lac ) in tandem form O), Bacillus subtilis shared ribosome binding site (RBS), ATG start codon, DNA sequence encoding this tiGH and DNA oligonucleotide sequence encoding 6 histidine residues (6x His tag) -Insert the DNA expression cassette into the shuttle vector of Bacillus subtilis or E. coli to form a recombinant construct 3vegAtiGHH6;-Transform the recombinant construct into the Bacillus subtilis host;-Make the subtilis The Bacillus host grows in cell culture and expresses the tiGH in the cell; and—using a French press homogenizer (French press homogenizer) to mechanically dissolve the Bacillus subtilis to release the tiGH.

According to the second aspect of the present invention, there is provided a method for expressing a gene encoding fish growth hormone (tiGH) of tilapia hormonalis in a Bacillus subtilis host, the method comprising the following steps:-preparing a recombinant DNA expression cassette, The DNA expression cassette includes: at least a pair of veg A promoter ( veg A) and lac operator ( lac O), Bacillus subtilis shared ribosome binding site, ATG start codon, DNA sequence encoding the tiGH, and DNA oligonucleotide sequence encoding 6 histidine residues (6x His tag);-insert the DNA expression cassette into the shuttle vector of Bacillus subtilis or Escherichia coli to form a recombinant construct 3vegAtiGHH6; The recombinant construct is transformed into the Bacillus subtilis host;-the Bacillus subtilis host is grown in cell culture and the tiGH is expressed in the cell; and-the tiGH is released.

Preferably, tiGH may be a single polypeptide of 22 kDa. The recombinant DNA expression cassette from 5'to 3'can include: three pairs of veg A promoter ( veg A) and lac operator ( lac O) in tandem, and Bacillus subtilis shared ribosome binding site , ATG start codon, the DNA sequence encoding the tiGH and the DNA oligonucleotide sequence encoding 6 histidine residues (6x His tag). The shuttle vector may have pMTLBs72 with a size of 6.62 kb. In the presence of IPTG inducers and/or chloramphenicol antibiotics, the transformed Bacillus subtilis host can be grown until the late logarithmic or stable phase.

In some embodiments, tiGH can be released from the Bacillus subtilis host mechanically, for example, by using a French pressure cell homogenizer. The method may include the step of centrifuging the cells of the Bacillus subtilis host to obtain tiGH.

According to the third party of the present invention, a method of preparing fish feed is provided, which includes the above-mentioned method of expressing the gene encoding the fish growth hormone (tiGH) of tilapia in a Bacillus subtilis host.

Preferably, the cell lysate produced by cell lysis may be coated on commercially available fish feed pellets or otherwise mixed in commercially available fish feed pellets.

In some embodiments, the method may include the step of preparing a solution using an agar solution containing substantially 55.56 µl of the cell lysate per ml, wherein the cell lysate may be prepared from substantially 1.112 ml of cell culture. The cell culture prepares cell lysates. The agar solution may substantially contain 0.5% sucrose and 1% agar, and may be prepared substantially at a temperature of 50°C. The mixed particles can then be dried at room temperature. The fish feed may basically include 1 µg of the tiGH/1 g feed.

According to the fourth aspect of the present invention, an engineered plasmid is provided, which contains a coding sequence, wherein the coding sequence includes the following:-a) at least a pair of veg A promoter ( veg A) and lac operator ( lac O) ); b) Shared ribosome binding site (RBS); c) Gene encoding tilapia growth hormone (tiGH); and d) Sequence encoding 6-histidine codon (6x His tag).

Preferably, the plasmid may contain at least three pairs of veg A promoter ( veg A) and lac operator ( lac O) operably linked from 5'end to 3'end . tiGH can be the growth hormone of tilapia. tiGH is operably linked to the 6-histidine codon at the 3'end of the coding sequence.

According to the fifth aspect of the present invention, there is provided a recombinant protein expressed by a host integrated with the plasmid. Preferably, the protein can be expressed in Bacillus subtilis.

According to the sixth aspect of the present invention, a recombinant Bacillus subtilis ( Bacillus subtilis ) containing the above-mentioned engineered plasmid is provided. Recombinant Bacillus subtilis can be configured to produce genetically engineered animal food or feed additives. Recombinant Bacillus subtilis can be configured to facilitate the detection and purification of tiGH expressed by it.

According to the seventh aspect of the present invention, there is provided an animal food or feed additive, which contains the recombinant Bacillus subtilis cell, its lysate and/or the protein expressed therefrom. The animal food or feed additive can be fish food or fish food additive, respectively. By coating the cells, lysate and/or protein, the animal food or feed additive can contain the cells, lysate and/or protein. The animal food or feed additive may contain the lysate of the culture in which the recombinant Bacillus subtilis and the agar solution melted at a ratio of substantially 1:18, wherein the melted agar solution comprises substantially 0.5% (w/v )sucrose. Animal food or feed additives can be configured to feed Japanese koi (Japanese koi) to improve their growth performance.

According to the eighth aspect of the present invention, there is provided a method for preparing recombinant Bacillus subtilis, the method including the step of integrating the above-mentioned plasmid into wild-type Bacillus subtilis.

According to a ninth aspect of the present invention, there is provided a method for preparing a culture lysate, the method comprising the step of preparing a lysate from the culture, in which the above-mentioned recombinant Bacillus subtilis grows.

According to the tenth aspect of the present invention, there is provided a method for increasing the growth rate of fish, the method including the step of feeding the fish with the animal feed or feed additive. Growth rate can include total body weight or total body length or both. The fish can be Japanese koi.

The present invention relates to the administration of fish growth hormone (FGH) to increase the growth rate of fish. For different reasons, Tilapia growth hormone (tiGH) is one of the focuses of the present invention. The research leading to the present invention shows that the recombinant form of tiGH can not only promote the growth of the bony fish species of tilapia, but also promote the growth of other fish, such as sockeye salmon, tilapia, goldfish, and trout larvae. , And larvae of carp and angelfish. More specifically, experiments during the process of the present invention proved that the engineered Bacillus subtilis (ie, GRAS) system can effectively and efficiently express active and functional recombinant tiGH (rtiGH) in the cytoplasm of the Bacillus subtilis host, It contains a 6-histidine tag (rtiGH) at its C-terminus. Experiments were performed to confirm the identity of the rtiGH produced, especially by Western blot analysis and mass spectrometry. The present invention also proposes a simple method for preparing fish feed by using rtiGH produced by the whole cell lysate of Bacillus subtilis containing rtiGH. A randomized controlled study was conducted to determine the effectiveness of supplementary feed and normal feed in promoting fish growth. Japanese koi was used as an example of a fish model. During the four-month study period, the weight and length of fish between the treatment group (40.38g and 2.17cm, respectively) and the control group (31.58g and 1.61cm, respectively) were compared. The conclusion is that the growth of fish in the treatment group was significantly faster than that in the control group. The fish fed the supplementary feed showed that they were healthy and well-developed at all times, active and eager to eat. See the example in Figure 10.

The following description and experiment prove the present invention in more detail. Materials and methods Bacterial strains and media

The Bacillus subtilis strain 1A751 ( eglS D102, bglT / bglS DEV, npr , apr , his ) used as a host for rtiGH expression was obtained from the Bacillus Genetics Stock Centre, Ohio ( Bacillus Genetics Stock Centre, Ohio). The E. coli strain JM101 ( supE , thi , D lac-proAB (F', tra 36, pro AB+, lacI ^q ZDM15)) used as an intermediate host for recombinant DNA work is as described above.

Bacillus subtilis transformants in 2x LB medium supplemented with 10.5 mg l ^-1 chloramphenicol (Fluka) at 37°C (20 gl ^-1 Difco trypsin, 10 gl ^-1 Difco yeast extract, 20 gl ^-1 NaCl) Medium growth. For solid media, add Bacto agar at a concentration of 1.5% (w/v). The calcium chloride method and the method described by Spizizen were used to transform Escherichia coli and Bacillus subtilis using recombinant plasmids, respectively. Species and sources of fish

(From Mermaid Koi Center, Hong Kong) 18 Japanese koi larvae (18-25 cm long; most: red and white), 6-8 months old, were purchased to set up negative experiments. On the other hand, 44 10-month-old Japanese koi carp larvae (mainly red and white) were kindly donated by East River Fishing Limited (Guangdong Province, China) to evaluate the biological activity of rtiGH as a randomized control the study. In addition, 8 koi fishes (7 of them 10 months old; 1 of 22 months old) were individually fed in a glass tank (150 cm×43 cm×63 cm) and fed with feed supplemented with rtiGH. Monitor their behavior and external characteristics within 5 months. Expression of plasmid and recombinant tiGH

The 6.62-kb Bacillus subtilis/E. coli shuttle vector pMTLBs72 was obtained from the Bacillus genetic bank center. The plasmid pMTLBs72 consists of a part of the Bacillus subtilis pBS72 plasmid and a part of the E. coli pBR322 sequence. It is a low copy number and stable plasmid in Bacillus subtilis, with about 6 copies/chromosome. Using pMTLBs72 as a vector (unpublished information), the modified plasmid 3vegAcenA was constructed as before. Using 3vegAcenA, the plasmid 3vegAtiGHH6 was constructed as follows. Using 14 oligomer primers: P1-P14 (please refer to the sequence listing shown as SEQ ID NOs. 1-14), the intermediate was synthesized by PCR overlap extension; the resulting product was cleaved with Spe I and Sma I, and then combined with The vector 3vegAcenA restricted by the same two enzymes was ligated to form 3vegAtiGHH6. Please refer to Figure 1.

In order to express rtiGH, fresh Bacillus subtilis 1A751 (3vegAtiGHH6) transformants were grown in 50 ml of 2x LB medium supplemented with chloramphenicol at 37°C for 8 hours. All cultures were then inoculated into 500 ml 2x LB medium with chloramphenicol and grown at 37°C until the A ₆₀₀ value was equal to 1.0, followed by the addition of IPTG to a final concentration of 0.1 mM. Continue cell culture for a period of 20 hours. Then, the culture was centrifuged and the cell mass was lysed. Cell lysis

The retained cell mass was dissolved in 25ml phosphate buffer (PB; 1.44 gl ^-1 Na ₂ HPO ₄ and 0.24 gl ^-1 KH ₂ PO ₄ ; pH 7.4), and a pressure cell homogenizer (Stansted Fluid Power Ltd., UK) Process the resuspension 3 times. Collect the lysate for analysis and feed preparation. Analysis of rtiGH

Protein samples separated on a 12% (w/v) tricine-SDS-polyacrylamide gel (tricine-SDS-polyacrylamide gel) were stained with Coomassie Brilliant Blue R250. Western blot analysis of protein was performed using His-probe antibody (H-3) (Santa Cruz, sc-8036). The identity of rtiGH was confirmed by liquid chromatography tandem mass spectrometry with Mascot search as described previously. Preparation of fish feed supplemented with lysate of bacterial culture

In a short time of only 20 seconds, 0.9 ml of agar solution (0.5% sucrose, 1% agar; 50°C) and 50 µl of cell lysate mixture containing rtiGH (concentration 100 mg l ^-1 ) showed that the mixture was uniformly and well Adsorbed on 5 g fish feed (Aqua master, Koi Food, Wheat Germ, size S).

To prepare fish feed supplemented with rtiGH, it was determined that 1 g of feed contained 1 µg rtiGH; therefore, 50 µl rtiGH lysate was used to mix with 0.9 ml agar solution. It was determined that the lysate of the induced 1A751 (3vegAtiGHH6) culture contained 100 mg l ^-1 rtiGH. It was determined that the protein concentration of the 1A751 (3vegAtiGHH6) lysate was 9.93 gl ^-1 , and the protein concentration of the host lysate (negative control) was 9.78 gl ^-1 . Since the same concentration of lysate protein was used to prepare various feeds, 1 g of control feed was calibrated to contain 10 µl of host lysate. The schematic details of the preparation of the feed supplemented with the culture lysate are described in Figure 2. Finally, the coated feed was air dried at room temperature for 3 days. Evaluation of the biological activity of rtiGH

Two gray plastic tanks sharing the same size (168 cm×64 cm×16 cm) were used to feed the two groups (treatment group and control group) fish. All variables in the two slots remain the same. Use a recirculation system with Qmax set to 2,200 lh ^-1 to facilitate water exchange in it. The water conditions in the two tanks were kept constant as follows: the temperature was 23°C, the pH was 6, and the dissolved oxygen was 6.3 mg l ^-1 .

In a negative experiment, the fish were fed normal food particles or food particles supplemented with Bacillus subtilis cell lysates lacking rtiGH activity. In a randomized controlled study, fish were fed normal food particles (control group) or food particles supplemented with cell lysates containing rtiGH (treatment group). At predetermined time intervals, each fish sample was photographed (for identification) and weighed, and its length was measured. Results The expression of tilapia growth hormone in Bacillus subtilis

The DNA sequence encoding tilapia growth hormone (tiGH) obtained by gene assembly was cloned into the 3VegAcenA vector (unpublished information) to form the rtiGH-expressing construct 3VegAtiGHH6 (see Figure 1). Under the induction of IPTG, Bacillus subtilis 1A751 (3VegAtiGHH6) cultures showed a high level of expression of intracellular rtiGH. When the cell lysate of 1A751 (3VegAtiGHH6) was analyzed by SDS-PAGE, it showed a clear band of rtiGH (22.6 kDa) with the expected size (see Figure 3). Subsequently, the band was confirmed to be tiGH by mass spectrometry (data not shown). The production of rtiGH is estimated to be about 100 mg l ^-1 . Preparation of fish food particles coated with Bacillus subtilis cell lysate

The conventional use of food pellets added with fish growth hormone (FGH) is not new. One novelty of the present invention relates to the specific application of the Bacillus subtilis lysate to the preparation of fish feed. Although considered GRAS, the Bacillus subtilis culture has an offensive smell. In order to solve the problem that the lysate of Bacillus subtilis has an unpopular odor for fish, another aspect of the present invention relates to an effective method for tightly coating rtiGH suspended in the cell lysate on food particles.

The good recovery and complete bands of rtiGH shown on the gel (see Figure 3) support the conclusion that high-pressure cell lysis is a practical method. In order to overcome the unpleasant smell of the lysate of Bacillus subtilis and the low efficiency of coating the lysate on the particles, the processability of soft agar and low-level sucrose was solved. Using this process, the bacterial lysate is uniformly and well coated on the particles (see Figure 2). Choose Japanese Koi as the fish model

Japanese koi (koi) is one of the most popular ornamental fish in China and Japan. Based on the following considerations, Koi is used as a fish model in the study. Koi are easy to obtain, are not picky about food, grow fast, and are basically raised for viewing.

When feeding koi with food pellets coated with Bacillus subtilis lysate and food pellets coated with Bacillus subtilis lysate containing rtiGH, the fish seemed to enjoy these two types of feed, and the behavior was similar to normal food pellets. the same. The effect of Bacillus subtilis lysate on the growth of Japanese koi

In order to determine whether the cell lysate of Bacillus subtilis lacking rtiGH may cause any stimulating effect on the growth of koi, firstly, the transformant Bacillus subtilis 1A751 (3VegA) containing only the 3VegA expression vector (data not shown) was obtained. A culture lysate of Bacillus subtilis 1A751 (3VegA) was then prepared and used for coating on food particles, thereby generating a negative control (untreated particles) for comparison. The 18 Japanese koi carps were divided into two groups, one group was fed untreated pellets and the other group was fed normal food pellets. The feeding regimen of the two groups of fish (twice/day and 0.5 g/fish) was the same, and the study lasted for 50 days. At the end of the study, the changes in the total weight and length of the fish in 50 days were evaluated (data not shown), and there was no significant difference in growth performance. Then it was concluded that the lysate of Bacillus subtilis lacking rtiGH activity had an effect on Japanese koi. Growth is not stimulating. Growth stimulating activity of rtiGH

The growth stimulating effect of rtiGH was determined by the dietary supply of the following two groups of koi. The treatment group was fed food pellets coated with the lysate of Bacillus subtilis 1A751 (3VegAtiGHH6) culture, while the control group was fed normal pellets. The growth parameters (weight and length) of each individual koi are measured at monthly intervals (see Figures 4 and 7). In the first two months of the study, the weight loss of most individuals in the two groups (see Figure 4) may be the result of fish’s negative reactions to various environmental impacts. Later, the weight and length measurements of almost all fish in the two groups recorded increased. By the end of the 4-month experiment, the increase in weight and length of the individuals in the treatment group showed significantly better performance than the corresponding individuals in the control group (see Figures 5 and 8). The overall average increase in weight of the treatment group was 27.9%, which was higher than that of the control group (see Figure 6), while the overall average increase in the length of the former was 34.8%, which was higher than the latter (see Figure 9). External view of Japanese koi fed with food pellets supplemented with rtiGH ( external view )

In order to get some views on the behavior and external characteristics of Japanese koi (treatment group) fed food pellets supplemented with the lysate of Bacillus subtilis containing rtiGH, eight were isolated from the second batch of fish (materials and methods) immediately after arrival. Individuals. These fish were then individually fed (materials and methods) in a glass tank, and basically the same way as the treatment group, only fed with the feed supplemented with rtiGH.

After 5 months of feeding, all fish appeared to be healthy, fleshy (and a few may even be considered stubby), sensitive and clear in color. The fish is not afraid of observers standing nearby, and actively swims around the tank. More importantly, they seem to be hungry at all times, and they maintain a high level of vigilance for food supplies whenever someone passes by the sink.

The availability of fish feed with high protein content is the main cost factor considered in fish farming. However, because of the high feed conversion efficiency (FCE) in fish farming; for example, in the process of feeding salmon, it is estimated that FCE is in the range of 1.1 kg feed/kg salmon. Therefore, in order to improve the growth performance of fish, a good idea that has flashed in mind is to increase the protein content of fish feed to increase FCE. Common ingredients used to increase protein content in fish feed include fish protein hydrolysate, brine shrimp, squid offal, soybeans, and blue-green seaweed.

In scientific efforts along the same research line, recent developments in biotechnology have enabled the development of genetically modified fish or genetically modified (GM) fish, in which the fish species of interest have been genetically mutated, resulting in excessive endogenous growth hormone Produced to increase the growth rate of fish. Although the FDA approved the use of GM fish for human consumption, this issue has been controversial. A good compromise on the use of fish growth hormone (FGH) seems to be its use as a feed supplement in the fish diet.

Recombinant FGH (rFGH) of different fish species has been successfully expressed and introduced in a variety of ways, including injection, bathing, oral incubation and feeding to fish models, to evaluate their growth-promoting performance under established conditions. However, in real situations, since fish farming involves large-scale farming, the first three methods seem impractical. Regarding the fourth method, feeding (referring to feeding fish with food pellets supplemented with rFGH) is technically feasible, as long as a cost-effective way of producing the feed of interest is available. Previous reports indicate that fish feed supplemented with freeze-dried Synechocystis (a kind of blue-green seaweed) expressing rFGH of olive flounder promotes the growth of flounder fish. However, although feed has a positive effect on growth performance, the report failed to address the potential harm to human health caused by the toxin Microcystin present in Synechocystis. This important problem, combined with the relatively long time required to prepare the final additive, presents a difficult challenge to the wide application of this feeding method in fish farming.

In contrast, with respect to the present invention, Bacillus subtilis, the host for rFGH expression, is recognized as a "GRAS" organism application. In addition, its well-characterized genetics, relatively short doubling time (120 minutes), easy and simple processing, and most importantly, convenient and cost-effective production scale-up support the use of Bacillus subtilis as a host Feasibility for restructuring applications. In fact, the method of preparing fish feed supplemented with the whole cell lysate of Bacillus subtilis expressing rtiGH (see Figure 2; materials and methods) is not only convenient but also easy to scale up. Regardless of whether the supply is small-scale or large-scale, supplemental feed, like its normal counterpart, facilitates distribution and access to the fish being fed.

Before the experiment, a relatively long period of four months (excluding the first two months spent on the negative experiment) was planned for the experiment. It was subsequently determined that the fish under observation might show uncomfortable reactions due to the new environment and measurement operations, which affected their appetite and led to misinterpretations of the conclusions drawn between diet composition and growth performance in the study. As predicted, most individuals in the treatment group (fed with rtiGH-supplemented feed; see Figure 4A) and control group (fed with normal feed; see Figure 4B) appeared to exhibit new arrivals and/or subsequent measurements of them Determine the unexpected reaction. Interestingly, these side effects only affect the weight of the two groups of fish (see Figure 4), but not the length (see Figure 7). In addition, in the first and second months of the study, the fish showed the most significant weight loss. Later, the fish seemed to adapt to their environment and regained interest in food, thus allowing us to draw a correlation between feed consumption and the increase in fish weight/length.

The results of the above discussion support the following conclusions. First, the two groups of koi fish consume a certain period of time to adapt to the environment and/or the operating process of introducing them newly. Second, each fish reacts differently to the new environment and/or new operation process. Therefore, the response of individual fish needs to be monitored. Third, due to the time required for adaptation, it was difficult to arrive at a reliable correlation between feed consumption and fish weight/length increase during the first two months of the study. Fourth, the body lengths of the two groups of fish seem to be less sensitive to the response to the environment. However, in the third and fourth months of the study, the changes in length and weight were closely aligned with each other, so this allowed us to draw a correlation between feed consumption and the increase in fish weight/length.

In the first two months of the study, regardless of the possible presence of environmental factors that may trigger weight loss in both the treatment group and the control group (see Figure 3), the difference in the overall average increase in body weight and length of the two groups of fish provides The decisive supporting evidence distinguishing the performance between normal fish feed and feed supplemented with rtiGH. Although the members used in the two groups of fish showed heavier weight and longer length after four months of research (see Figures 5 and 8), the overall average increase in weight and length of the treatment group was 40.38, respectively. g (see Figure 6) and 2.17cm (see Figure 9), which are significantly higher than the untreated group's values, respectively 31.58g (see Figure 6) and 1.61cm (see Figure 9). In fact, the discrepancies between the two sets of data are significantly different, with an overall average increase in weight of 27.9% (see Figure 6) and an overall average increase of 34.8% in length (see Figure 9).

Therefore, our results provided in this application support the conclusion that rtiGH expressed in Bacillus subtilis has biological activity in promoting the growth of Japanese koi fish. In addition, the convenience of obtaining a whole cell lysate of Bacillus subtilis provides a simple means for preparing fish feed supplemented with rtiGH. Continuing efforts to improve tiGH expression, protein content and growth density of Bacillus subtilis, as well as conditions for scale-up production, this method can prove to be a cost-effective method for large-scale application in fish farming.

Growth rate is one of the most important points considered by fisheries, because there is a direct relationship between growth performance and profit yield. The fish growth hormone (FGH) produced by the pituitary gland, a 22 kDa single chain polypeptide, not only has a positive effect on growth, but also has a pleiotropic function, which is better for fish under adverse conditions (such as environmental pressure and infection) Coping is key.

Many species of FGH have been well characterized, and their corresponding recombinants have been expressed in different microbial host systems for research. Among the various FGHs, Tilapia, one of the adult tilapias that can reproduce in seawater and freshwater, is one of the focuses of the present invention. The natural FGH of tilapia (tiGH) is a polypeptide with 187 amino acids, which has a high level of homology (over 84%) with the FGH of tuna and yellowtail, and is 66% identical to trout and salmon. TiGH is also expressed in E. coli and Pichia pastoris in recombinant form. Due to its improved availability, the effectiveness of recombinant tiGH in affecting the growth rate of fish has been fully studied. In one example, although the tiGH and FGH of trout and salmon have a relatively low level (66%) of identity, there are reports that the purified recombinant tiGH from E. coli not only promotes the bony fish of tilapia The species Oreochromis mossambicus grows, and it also promotes the growth of sockeye salmon Oncorhynchus nerka . In another embodiment, the recombinant tiGH prepared by Pichia pastoris showed enhanced growth, survival and quality of larvae and immunity of several larvae and larvae, such as tilapia, goldfish and trout larvae and carp And larvae of angel fish.

Interestingly, unlike the corresponding mammals, it has been shown that FGH can be administered orally to improve the growth performance of fish. This strategy works well in bony fish because their intestinal epithelium can absorb high molecular weight proteins. As expected, biologically active tiGH has been shown to be well absorbed by fish intestines to promote physical growth. In addition, oral administration of FGH is related to the growth promotion of other species of fish, including coho salmon (coho salmon), flounder ( Paralichtys olivaceus ), sea bass ( Perca fiuviatilis ) and giant catfish (giant catfish). More importantly, no trace of FGH administered orally was detected in the blood, intestine, muscle or liver of the fish 90 minutes after administration. Therefore, oral administration of FGH seems to provide a safe way to improve the growth performance of fish.

One way to treat fish samples with FGH is by soaking the fish in a buffer solution in which FGH is dissolved. Although the treated fish grow significantly faster than the control fish soaked in a common buffer solution, this method of administration is inefficient, labor-intensive and not feasible on a large scale.

Our laboratory has been involved in the construction of an effective microbial system for the production of recombinant proteins for commercial use. In addition to the well-characterized gram-negative bacterium Escherichia coli, we believe that the gram-positive bacterium Bacillus subtilis, which has not been fully studied like Escherichia coli, has great development potential as a universal recombinant host system. Due to the lack of an external membrane, Bacillus subtilis is well suited for engineering to secrete and produce foreign proteins. In addition, due to the lack of an outer membrane, Bacillus subtilis does not contain endotoxins, so its use is generally considered safe (GRAS).

The present invention pioneered the development of a Bacillus subtilis secretion system for the recombinant production of beneficial proteins, including human epidermal growth factor and endoglucanase from cellulolytic bacteria ( Cellulomonas fimi ). Subsequently, derivatives of the veg I promoter of Bacillus subtilis were constructed and proved to be functionally active in E. coli.

It should be understood that, for the sake of clarity, certain features of the present invention described in separate embodiments or contents of experiments may be provided in combination in a single embodiment. On the contrary, for the sake of brevity, the various features of the present invention described in the content of a single embodiment may be provided individually or in any suitable sub-combination. It should be noted that certain features of the embodiments are illustrated by non-limiting examples. Also, those skilled in the art are aware of the prior art that is not explained above for the sake of brevity. Some of the prior art techniques are listed below, which are incorporated herein in their entirety by reference.

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Aquacult Res 1-6 Doi:10.1111/j.1365-2109.2012.03168. x Hertz Y, Tchelet A, Madar Z, et al. (1991) Absorption of bioactive human growth hormone after oral administration in the common carp ( Cyprinus carpio ) and its enhancement by deoxycholate. J Comp Physiol B 161(2): 159-163. Liu S, Zhang X, Zang X et al. (2008) Growth, feed efficiency, body muscle composition, and histology of flounder ( Paralichthys olivaceus ) fed GH transgenic Synechocystis . Aquaculture 277(1-2): 78-82. Vareli K, Jaeger W, Touka A and others (2013) Hepatotoxic Seafood Poisoning (HSP) Due to Microcystins: A Threat from the Ocean? Marine Drugs 11: 2751-2768. Olmos J and Paniagua-Michel J. (2014) Bacillus subtilis a potential probiotic bacterium to formulate functional feeds for aquaculture . J Microb Biochem Technol 6(7): 361-365.

lac O‧‧‧ lac operator RBS‧‧‧shares ribosome binding site tiGH‧‧‧fish growth hormone veg A‧‧‧ veg A promoter

The details and some embodiments of the present invention will now be explained with reference to the drawings, in which: Figure 1 is a schematic diagram showing an embodiment of the recombinant DNA construct according to the present invention; Figure 2, including A and B, is a diagram showing the basis of the present invention. Photographic images and diagrams of an embodiment of the method for preparing fish feed or feed additives of the present invention; Figure 3, including A and B, is an image of Western blot analysis of an exemplary Bacillus subtilis cell lysate obtained according to the present invention Figure 4, including A and B, is a diagram showing the experimental results of fish treated with food containing and without recombinant growth hormone prepared according to an embodiment of the present invention; Figure 5, including A and B, is A graph showing the growth of fish treated with and without using the method or means according to an embodiment of the present invention; Figure 6 is a graph showing two groups of fish treated with and without using the method and means for growth of the present invention, respectively A graph of the overall average increase in weight; Figure 7, including A and B, is a graph showing the effect of adding growth hormone feed pellets according to an embodiment of the present invention; Figure 8, including A and B, is a display A graph showing the effects of using and not using a fish feed or fish additive treatment according to one embodiment of the present invention on the body length of fish; Figure 9 is a diagram showing the two treatments using and not using the method or means for growth according to the present invention, respectively The graph showing the overall average increase in the length of the group fish; and graph 10 shows the effects of growth hormone on tilapia, trout, goldfish, and scalar, respectively. Comparison chart of processing results.

＜110＞ NG, KA LUN ALAN ＜120＞ MEANS AND METHOD FOR ENHANCING GROWTH OF FISH ＜130＞ G02-006A ＜140＞ ＜141＞ ＜150＞ HK 15112136.3 ＜151＞ 2015-12-09 ＜160＞ 14 ＜170 > PatentIn version 3.5 <210> 1 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 1 acactagtaa aaagatatct aatttaaatt ttatttgaca aaaatgggct cgtgttgtac 60 aat 63 <210> 2 ＜211＞ 60 ＜212＞ DNA ＜213＞ Artificial Sequence ＜220＞ ＜223＞ Description of Artificial Sequence: Synthetic primer ＜400＞ 2 gaacaaacgc tggctgtctg tgatctgctg caattttatc acctcctttg tgaaattgtt＞ 60 212 212 DNA ＜213＞ Artificial Sequence ＜220＞ ＜223＞ Description of Artificial Sequence: Synthetic primer ＜400＞ 3 aacaatttca caaaggaggt gataaaattg cagcagatca cagacagcca gcgtttgttc＜213 212＞ 4 ＜211＞ 75 ＜212＞ 223＞ Description of Artif icial Sequence: Synthetic primer <400> 4 cagagagctc tcaaagtccg agaagagtct ctgggcgagc aggtgcaggt gcgtgactct 60 gttgactgca atgga 75 <210> 5 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer < 400> 5 cttctcggac tttgagagct ctctgcagac ggaggagcaa cgtcagctca acaaaatctt 60 cctgcaggac ttctg 75 <210> 6 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 6 tgcgctgcgt ctcgtgtttg tcgatcgggc tgatgatgta atcagagttg cagaagtcct 60 gcaggaagat tttgt 75 <210> 7 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 7 atcgacaaac acgagacgca gcgcagctcg gtcctgaagc tgctgtcgat ctcctatgga 60 ctggttgagt cctgg 75 <210> 8 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 8 atctggttcc tcagagagga acctccagac agagagcgac tgggaaactc ccaggactca 60 accagtccat aggag 75 <210> 9 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 9 gaggttcctc tctgaggaac cagatttcac caaggctgtc tgagcttaaa acgggaatct 60 tgctgctgat caggg 75 <210> 10 <211> 75 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 10 gtgctggagg gtgtcggtgt caggataatt ctctgcttca tcctgattgg ccctgatcag 60 cagcaagatt cccgt 75 <210> 11 <211> 75 <212> DNA <213> Artific ial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 11 tcctgacacc gacaccctcc agcacgctcc ttacggaaac tattatcaaa gtctgggagg 60 caacgaatcg ctgag 75 <210> 12 <211> 75 <212> DNA <213> Artificial Sequence <220> < 223> Description of Artificial Sequence: Synthetic primer <400> 12 ccaccttgtg catgtccttc ttgaagcaag ccagcaattc ataagtttgt ctcagcgatt 60 cgttgcctcc cagac 75 <210> 13 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Synthetic primer <400> 13 ttcaagaagg acatgcacaa ggtggagacc tacctgacgg tagctaaatg tcgactctct 60 ccagaagcaa actgc 75 <210> 14 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 14 agccccgggc taatgatgat gatgatgatg cagagtgcag tttgcttctg gagagagtcg 60 a 60

lac O‧‧‧ lac operator

RBS‧‧‧ Shared ribosome binding site

veg A‧‧‧ veg A promoter

Claims (13)

A method for expressing a gene encoding fish growth hormone (tiGH) of Oreochromis hornorum in a Bacillus subtilis host, the method comprising the following steps:-Preparation of a recombinant DNA expression cassette, the DNA expression cassette is from 5 The'end to 3'end includes: three pairs of veg A promoter ( veg A) and lac operator ( lac O) in tandem, Bacillus subtilis shared ribosome binding site (RBS), ATG start code DNA sequence encoding the tiGH and a DNA oligonucleotide sequence encoding 6 histidine residues (6x His tag);-insert the DNA expression cassette into the shuttle vector of Bacillus subtilis or E. coli, thereby Forming the recombinant construct 3vegAtiGHH6;-transforming the recombinant construct into the Bacillus subtilis host;-allowing the Bacillus subtilis host to grow in cell culture and express the tiGH in the cell; and-by dissolving the Bacillus subtilis Release the tiGH. A method for expressing a gene encoding fish growth hormone (tiGH) of tilapia in Bacillus subtilis host, the method comprising the following steps:-preparing a recombinant DNA expression cassette, the DNA expression cassette comprising: at least a pair of veg A promoter ( veg A) and lac operator ( lac O), Bacillus subtilis shared ribosome binding site, ATG start codon, the DNA sequence encoding the tiGH and the 6 histidine residues (6x His tag) DNA oligonucleotide sequence;-insert the DNA expression cassette into the shuttle vector of Bacillus subtilis or E. coli to form the recombinant construct 3vegAtiGHH6;-transform the recombinant construct into the Bacillus subtilis host Medium;-growing the Bacillus subtilis host in cell culture and expressing the tiGH in the cell; and-releasing the tiGH. The method described in item 2 of the scope of patent application, wherein the tiGH is a single polypeptide of 22 kDa. The method according to item 2 or item 3 of the scope of patent application, wherein the recombinant DNA expression cassette includes from 5'end to 3'end : three pairs of veg A promoters ( veg A) and lac operating gene ( lac O), Bacillus subtilis shared ribosome binding site, ATG start codon, DNA sequence encoding the tiGH and DNA oligomers encoding 6 histidine residues (6x His tag) Nucleotide sequence. The method described in item 2 or item 3 of the scope of patent application, wherein the shuttle vector is pMTLBs72 with a size of 6.62 kb. The method according to item 2 or item 3 of the scope of patent application, wherein the transformed Bacillus subtilis host is grown in the presence of an IPTG inducer and/or a chloramphenicol antibiotic until the late logarithmic or stable phase. According to the method described in item 2 or item 3 of the scope of patent application, the method includes the step of centrifuging the cells of the Bacillus subtilis host to obtain the tiGH. The method described in item 1, item 2 or item 3 of the scope of the patent application, wherein the release of tiGH is achieved mechanically by using a French pressure cell homogenizer. A method for preparing fish feed, which includes the method of expressing the gene encoding fish growth hormone (tiGH) of tilapia hormonalis in the Bacillus subtilis host described in item 1 of the scope of patent application, wherein tiGH is released The steps include mechanically dissolving the Bacillus subtilis host to produce a cell lysate containing tiGH, and coating the fish feed pellet with the cell lysate containing tiGH. An engineered plasmid containing: coding sequences with at least a pair of veg A promoter ( veg A) and lac operator ( lac O), shared ribosome binding site (RBS), coding for tilapia growth hormone The gene of (tiGH) and the sequence encoding the 6-histidine codon (6x His tag), wherein the plasmid also contains at least three pairs of veg A promoters operably linked from 5'end to 3'end ( veg A) and lac operator ( lac O). A recombinant Bacillus subtilis containing the engineering plasmid described in item 10 of the scope of patent application. An animal food or feed additive, which contains the recombinant Bacillus subtilis cell, its lysate, or the protein expressed by the recombinant Bacillus subtilis described in item 11 of the scope of patent application. A method for preparing recombinant Bacillus subtilis, which includes the step of integrating an engineered plasmid into a wild-type Bacillus subtilis to produce a protein. The plasmid contains a coding sequence with at least a pair of vegA promoter (vegA) and lac operator (lacO); consensus ribosome binding site (RBS); gene encoding tilapia growth hormone (tiGH); and sequence encoding 6-histidine codon (6x His tag).

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